We evaluated an autoradiographic (ARG) method to calculate regional cerebral blood flow (rCBF) sequentially before and after an acetazolamide (ACZ) challenge in a single session of single-photon emission tomography (SPET) with two injections of N-isopropyl-[(123)I] p-iodoamphetamine (IMP). The method uses a table look-up method with a fixed distribution volume (Vd) and a standard input function of IMP. To calculate rCBF after an ACZ challenge, two look-up tables (a dual-table) are used to reflect the effect of radioactivity in the brain from the first dose of IMP. We performed simulation studies to evaluate errors attributable to (a) a change in rCBF induced by an ACZ challenge during the scan and (b) a fixed Vd value that might be different from an individual one, along with the effect of (c) scan length. Thirty-three patients were studied by dynamic SPET with two injections of IMP and frequent arterial blood sampling, and the data were analysed using the dual-table ARG method. Twenty-four of the 33 patients received an injection of ACZ 10 min before the second dose of IMP. We generated a standard input function by averaging individual input functions. The optimal method to calibrate a standard input function was determined so that the SD of differences between rCBF calculated by using a calibrated standard input function (F(SIF)) and that calculated by using an individual input function (F(IIF)) was minimised. Reliability of the method was evaluated by comparing F(SIF) with gold standard rCBF (F(REF)) obtained by two-compartment model analysis of dynamic SPET data and an individual input function with a non-linear least squares fitting method. Errors caused by (a) were less than 4% for a first rCBF ranging between 20 and 60 ml 100 g(-1) min(-1) and an rCBF change of between -25% and 50%. Errors caused by (b) were relatively large compared with those caused by (a), and were affected by (c) with an increasing error in a longer scan. In the patient study with a proposed scan protocol of 25 min for the first and 15 min for the second measurement, the error attributable to the standard input function was smaller when calibrated with a continuously drawn arterial blood sample (random error of 3.8% for continuous 10-min arterial blood sampling after the second dose of IMP) than with a single arterial blood sample (random error of 9.0% at 5 min after the second dose of IMP). Systematic and random errors of F(SIF) compared with F(REF) were 0.0% and 6.3%, respectively. The dual-table ARG method can be reliably used to quantify rCBF before and after an ACZ challenge with a 40-min scan protocol and continuous arterial blood sampling for several minutes.